I'm currently working on a project with two tumor cell lines each from a different patient, and each one of the tumor cell lines has a control and treated group. I'm trying to look at the changes in gene expression that occur in each tumor cell line between untreated and those treated with Treatment A using DESeq2, but i'm having trouble and keep getting a warning message when running the DESEq pipeline

>dds<-DESeq(dds)
estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing
-- standard model matrices are used for factors with two levels and an interaction,
where the main effects are for the reference level of other factors.
see the 'Interactions' section of the vignette for more details: vignette('DESeq2')

Is there another way to run the differential expression analysis to look at the change in expression level between the control and treated for each cell line ? I'm not sure why i keep getting this error. I've tried running DESeq2 with 1 cell line at a time (2 groups instead of 4) and I got very different results (only 5 genes instead of 81)

As for the design of the DESeqDataSet, I used design=~Cell_Treat, but it gave an error that the design has a single variable, with all samples having the same value and suggested I used a design of ~1.

I read the previous answer and tried to combine the variables in the dds, however i'm confused on the design set up for DESeqDataSet. I tried design=~Cell + Treatment and with that I was able to run DESeq, but when I tried to make a contrast of the groups I got an error that 'x' must be an array of at least two dimensions

Thanks! that helped a lot, i didn't notice I didn't specify the column from se and changed the code to colData(se)$Cell and the rest of the DESeq ran perfectly without any errors!

I contrasted the A1 vs A2 and B1 vs B2 and filtered out the results based on padj and log2FoldChange and found 1330 differentially expressed genes in both comparisons which I think makes sense because the drug must only effect certain genes when a cell is treated.